A homogeneous fluorescence polarization assay for detection of antibody to Brucella abortus

Abstract

A homogeneous fluorescence polarization (FP) assay (FPA) was developed for detection of antibody in bovine sera to Brucella abortus. The assay used O-polysaccharide prepared from B. abortus lipopolysaccharide in the molecular weight range of 20–30 kDa which was conjugated with fluorescein isothiocyanate and used as a tracer. Fluorescence polarization was measured with a FPM-1 fluorescence polarization analyzer. Sample (20 μl) was added to 2.0 ml of dilunet buffer at ambient temperature. A serum blank reading was taken and tracer (10 μl) to yield approx. 1.5 nM fluorescein equivalents was added. The FP of the tracer was determined after a period of greater than 2 min. A positive reaction was indicated by a significant elevation of the FP reading over the negative control. In a blind study, 9480 bovine sera were tested in addition to sets of four controls which were included with each lot of 100 samples tested. The controls were a strong positive, a weak positive, a negative and a serum derived from a B. abortus strain 19 vaccinated cow. Test sera included 8669 sera from Canadian cattle which were negative by routine serological tests, 561 sera from cows from which B. abortus had been isolated either from tissues or milk and 250 sera from cattle previously vaccinated with B. abortus strain 19 at various times. One lot of O-polysaccharide tracer was used for all tests. The initial cut-off for negative samples in the fluorescence polarization assay was set at 107.2 mP. This resulted in a sensitivity estimate of 98.1 ± 1.1% and the specificity was 99.8 ± 0.09%. After decoding the samples and retesting false positive and negative reactions, the sensitivity estimate was 98.5 ± 1.0% and the specificity was 100%. It became evident that the initial cut-off value was set too high and, using ROC analysis, a cut-off of 90 mP increased the sensitivity to 99.02% while the specificity decreased to 99.96%. Of the 250 sera from vaccinated cattle, 248 were negative giving a point specificity value of 99.2%.